Devices and methods for measuring nanometer level binding reactions

ABSTRACT

Systems and methods for measuring nanometer level binding reactions are described herein. In some embodiments, the disclosed subject matter is directed to a device comprising (a) a substrate having a surface and (b) an ordered array of posts, pits, or patches over the surface, wherein the posts, pits, or patches are capable of binding a protein or small molecule ligand, and wherein the pitch between adjacent posts, pits, or patches is less than about 100 nm. In some other embodiments, the disclosed subject matter is also directed to methods for identifying the presence of an analyte in a fluid and to methods for measuring relative binding specificity or affinity between an analyte in a fluid and the posts, pits, or patches, using the device of the disclosed subject matter.

This application claims the benefit of the filing date of InternationalApplication Serial No. PCT/US2004/034987, filed Oct. 15, 2004, and U.S.patent application Ser. No. 11/404,716, filed Apr. 13, 2006, both ofwhich claim the benefit of provisional application U.S. Ser. No.60/511,799, filed Oct. 15, 2003, which are hereby incorporated byreference into the subject application in their entireties. Thisapplication also claims the benefit of the filing date of provisionalapplication U.S. Ser. No. 60/837,701, filed Aug. 15, 2006, which ishereby incorporated by reference into the subject application in itsentirety.

All patents, patent applications and publications cited herein arehereby incorporated by reference in their entirety. The disclosures ofthese publications in their entireties are hereby incorporated byreference into this application in order to more fully describe thestate of the art as known to those skilled therein as of the date of thedisclosed subject matter described and claimed herein.

Copyright Statement: This patent disclosure may contain material that issubject to copyright protection. The copyright owner has no objection tothe facsimile reproduction by anyone of the patent document or thepatent disclosure as it appears in the U.S. Patent and Trademark Officepatent file or records, but otherwise reserves any and all copyrightrights.

TECHNOLOGY AREA

Devices and methods for measuring nanometer level binding reactions areprovided.

BACKGROUND OF THE APPLICATION

Many proteins have been studied extensively at the single moleculelevel. However, in the cell those proteins form into larger complexes ormodules wherein the spacing of components on a nanometer scale iscritical. New technologies in patterning now enable us to systematicallymeasure the dependence of interactions on nanometer level patterns andto then exploit that spatial dependence in sensing and nanofabricationof materials through directed self-assembly. As an example, the signalsfrom extracellular matrices affect normal and cancerous cell growth andthere is evidence that the spacing of the matrix molecules makes acritical difference in that signal (Jiang, G. et al. (2003) Nature,424:334-37). These mechanisms must be studied at a scale that matchesthe size and/or spacing of features of specific protein or subcellularprotein complexes, which are generally at the nanometer level.

There is a great need to measure the binding of complex proteinassemblies with spatially ordered ligands. Thus, there is a great needfor a device that tests the binding of an analyte to specific spatialarrays of ligands.

SUMMARY OF THE APPLICATION

In some embodiments, the disclosed subject matter is directed to adevice for measuring nanometer level binding reaction. The device caninclude (a) a surface and (b) an ordered array of posts, pits, orpatches on the surface, wherein the posts, pits, or patches are capableof binding a protein or small molecule ligand, and wherein the pitchbetween adjacent posts, pits, or patches is less than about 100 nm.

In some other embodiments, the disclosed subject matter is also directedto methods for measuring nanometer level binding reactions. Methods ofthe disclosed subject matter can include (a) providing a device having asurface, the surface comprising an ordered array of posts, pits, orpatches, wherein the pitch between adjacent posts, pits, or patches isless than about 100 nm, wherein each post, pit, or patch is coated withligand, (b) contacting the surface of the device with a sample, and (c)determining whether or not an analyte from the sample interacts or bindsto a ligand-coated post, pit, or patch, thereby identifying the presenceof the analyte in the fluid sample. The method can also includeisolating the analyte from the sample.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 depicts fibronectin and a slip bond formed between a singletrimer of fibronectin and a cellular cytoskeleton.

FIG. 2 depicts a proposed process flow for the fabrication of a deviceaccording to some embodiments of the disclosed subject matter.

FIG. 3 depicts features with nanometer scale dimensions patterned byelectron beam lithography.

FIG. 4 shows arrays of dots of hydrogen silsequioxane, a negative toneelectron beam resist.

FIG. 5 depicts an electrode pair for the study of transport inindividual molecules fabricated by direct-write e-beam lithography.

FIG. 6 is a graphic representation of resist thickness as a function ofapplied dose for a 3:1 isopropanol:water mixture using various molecularweights of poly(methyl)methacrylate.

FIG. 7 shows surface roughness of PMMA as a function of molecularweight.

FIG. 8 illustrates the reduction of dot size by thermal treatment.

FIG. 9 gives an example of high resolution placement accuracy e-beamlithography.

FIG. 10 depicts an exemplary device according to some embodiments of thedisclosed subject matter.

FIG. 11 depicts micrographs of a prototype chip with patterned arraysaccording to some embodiments of the disclosed subject matter.

FIG. 12 depicts prototype dot pair arrays according to some embodimentsof the disclosed subject matter.

FIG. 13 shows a prototype dot array according to some embodiments of thedisclosed subject matter.

FIG. 14 depicts atomic force microscopy of prototype arrays according tosome embodiments of the disclosed subject matter.

FIG. 15 shows a system for fabricating exemplary devices according tosome embodiments of the disclosed subject matter.

FIG. 16 shows lithographic ways of patterning according to someembodiments of the disclosed subject matter.

DETAILED DESCRIPTION OF THE APPLICATION

The present application enables the formation of protein arrays on thescale of nanometers as in a cell and the application of this technologyto measure and exploit the spatial dependence of interactions inbiological and nanofabrication areas. There is a need to control thespatial parameter in molecular interactions in a defined way.

The disclosed subject matter provides a device that facilitates bindingof an analyte to a set of posts, pits, or patches having a spacing thatfacilitates the binding. Binding to a single post, pit, or patch isdependent upon the chemistry and steric nature of the analyte and theprotein attached to the post, pit, or patch.

The device can include (a) a surface and (b) an ordered array of posts,pits, or patches on the surface, wherein the posts, pits, or patches arecapable of binding a protein or small molecule ligand, and wherein thepitch between adjacent posts, pits, or patches is less than about 100nm.

The disclosed subject matter is also directed to methods for measuringnanometer level binding reaction. Methods of the disclosed subjectmatter can include (a) providing a device having a surface, the surfacecomprising an ordered array of posts, pits, or patches, wherein thepitch between adjacent posts, pits, or patches is less than about 100nm, wherein each post, pit, or patch is coated with ligand, (b)contacting the surface of the device with a sample, and (c) determiningwhether or not an analyte from the sample interacts or binds to aligand-coated post, pit, or patch, thereby identifying the presence ofthe analyte in the fluid sample. The method can also include isolatingthe analyte from the sample.

The present application is further directed to methods for measuringrelative binding specificity or affinity between an analyte and aligand. Methods of the disclosed subject matter can include (a)providing a device having a surface, wherein the surface comprises anordered array of posts, pits, or patches on the surface having a ligand,wherein the pitch between adjacent posts, pits, or patches is less thanabout 100 nm, (b) contacting the surface of the device with one or moreanalytes, (c) determining whether or not an analyte interacts or bindsto the ligand, and (d) determining the binding specificity or affinitybetween the analyte and the ligand.

The disclosed subject matter is also directed to methods forcrystallizing a protein. Methods of the disclosed subject matter caninclude (a) providing a device having a surface, wherein the surfacecomprises an ordered array of posts, pits, or patches over the surface,wherein the pitch between adjacent posts, pits, or patches is less thanabout 100 nm, and is identical throughout the array, and wherein thepost, pit, or patch is capable of binding a protein, and (b) contactingthe surface of the device with a protein to be crystallized, wherein atleast one post, pit, or patch functions as a seed crystal or nucleus forcrystallization, thereby crystallizing the protein.

The disclosed subject matter is directed to methods for making a device.Methods of the disclosed subject matter can include (a) designing anarray pattern, (b) writing the array pattern onto a substrate, (c)forming a post, pit, or patch on the substrate, and optionally (d)shrinking or enlarging the post, pit, or patch, thereby making thedevice.

The disclosed subject matter is also directed to kits. Kits of thedisclosed subject matter can test for the presence or absence of ananalyte in a sample, determine a subject's risk for developing adisease, or monitor the status of a disease in a subject. Kits of thedisclosed subject matter can include a device that specifically binds toan analyte in an amount effective to detect the analyte in the sample.The kit can contain a device having a surface and an ordered array ofposts, pits, or patches over the surface, wherein the posts, pits, orpatches are capable of binding a ligand and wherein the pitch betweenadjacent posts, pits, or patches comprises less than about 100 nm.Further, the interaction between the device and the analyte isdetectable. The kit can include one or more reagents for detectingamounts of one or more analytes bound to the device. In anotherembodiment, the kit can further include one or more reagents fordetecting amounts of the one or more analytes bound to the device.

The disclosed subject matter is further directed to methods fordetecting a protein isomer in a mixture. Methods of the disclosedsubject matter can include (a) providing a device having a surface; thesurface comprising an ordered array of posts, pits, or patches over thesurface, wherein the pitch between adjacent posts, pits, or patches isless than about 100 nm, wherein the post, pit, or patch is coated withligand; (b) contacting the surface of the device with a mixture; and (c)determining whether or not a protein isomer in the mixture interacts orbinds to a ligand-coated post, pit, or patch, thereby detecting theprotein isomer in the mixture.

The disclosed subject matter is directed to methods for detecting amicroorganism in a sample. Methods of the disclosed subject matter caninclude (a) providing a device having a surface; the surface comprisingan ordered array of posts, pits, or patches over the surface, whereinthe pitch between adjacent posts, pits, or patches is less than about100 nm, wherein the post, pit, or patch is coated with ligand; (b)contacting the surface of the device with a sample; and (c) determiningwhether or not a microorganism in the sample interacts or binds to aligand-coated post, pit, or patch, thereby detecting the microorganismin the fluid sample.

In one embodiment, the device comprises a gradient of pitch values. Forexample, the gradient of pitch values can be from about 5 nm to about100 nm. In one embodiment, the pitch is less than about 50 nm. In otherembodiments, the pitch is less than about 40 nm, less than about 35 nm,less than about 30 nm, less than about 25 nm, less than about 20 nm,less than about 15 nm, less than about 10 nm, and in another embodiment,less than about 5 nm, less than about 3 nm in diameter, and in anotherembodiment, less than about 2 nm in diameter.

In another embodiment, the pitch is homogenous. For example, the orderedarray of pairs of posts, pits, or patches can have a pitch that is lessthan about 50 nm. In other embodiments, the pitch is less than about 40nm, less than about 35 nm, less than about 30 nm, less than about 25 nm,less than about 20 nm, less than about 15 nm, less than about 10 nm, andin another embodiment, less than about 5 nm, less than about 3 nm indiameter, and in another embodiment, less than about 2 nm in diameter.

In another embodiment, each post, pit, or patch is less than about 20 nmin diameter. In other embodiments, each post, pit, or patch is less thanabout 10 nm in diameter, less than about 5 nm in diameter, less thanabout 3 nm in diameter, and in another embodiment, less than about 2 nmin diameter.

In one embodiment, each post, pit, or patch independently comprises amaterial selected from the group consisting of metal, semiconductor,organic insulator, inorganic insulator, biocompatible material, or acombination thereof. For example, each post, pit, or patch independentlycomprises a material selected from gold, nickel, palladium,polyethyleneglycol, oxides of aluminum, hafnium, silicon, tantalum,titanium, zinc, or zirconium, nitrides of aluminum, hafnium, silicon,tantalum, titanium, zinc, or zirconium or carbides of aluminum, hafnium,silicon, tantalum, titanium, zinc, or zirconium or combinations thereof.In another embodiment, each post, pit, or patch includes a materialwhich does not quench fluorescence, such as titanium oxide. In yetanother embodiment, each post, pit, or patch includes a material thatprovides small grain structure and a smooth surface, such as agold-palladium alloy.

In another embodiment, each post, pit, or patch has affixed theretoexactly one protein molecule. In another embodiment, each post, pit, orpatch displays more than one protein molecule. In another embodiment,the protein molecule comprises at least a portion of a full-lengthdynein heavy chain, a tubulin, a kinesin, a myosin, a cytoplasmic domainof an integrin, an actin, an extracellular matrix protein, afibronectin, a collagen, a laminin, a DNA solenoid, a DNA, a histone-DNAcomplex, an RNA, an RNA-protein complex, a bacterial coat protein, anantibody, a lectin, an avidin, or any combination thereof. In oneembodiment, the protein molecule comprises a full-length dynein heavychain, a dynein motor domain, or an N-terminal portion of a dynein motordomain.

In one embodiment, the relative binding specificity or affinity is afunction of pitch. For example, pitch can be increased from a smallspacing to a large spacing to determine the maximum separation thatallows binding.

In one embodiment of the disclosed subject matter, the subject is amammal. The mammal can be a human or a primate. The subject can be ahuman patient, or an animal that exhibits symptoms of a human immunedisease and is therefore an animal model of a human disease, such as amurine transgenic disease model or a primate disease model or a model ofhuman disease established in a SCID mouse reconstituted with the humanimmune system. The mammal can be, but is not limited to, a human, aprimate, a rat, a dog, a cat, a swine. In another aspect of thedisclosed subject matter, the subject is a murine subject, a bovinesubject, a primate subject, an equine subject, a swine subject, or acanine subject.

In one embodiment, the sample is a blood sample or a serum sample.

In one embodiment, the analyte is labeled with a detectable marker. Inone embodiment, the detectable marker is selected from the groupconsisting of a fluorescent marker, a radioactive marker, an enzymaticmarker, a colorimetric marker, a chemiluminescent marker or acombination thereof.

In one embodiment, optical means, electrical means, mechanical means, ora combination thereof can be utilized to detect the presence of aspecies on a post, pit, or patch. In one embodiment, total internalreflection fluorescence (TIRF) microscopy, ellipsometry, or phasemicroscopy, atomic force microscopy (AFM), fluorescence resonance energytransfer (FRET) microscopy, fluorescence microscopy, two-photonmicroscopy, electrical conduction, or a combination thereof can beutilized.

In one embodiment, designing the array pattern comprises using acomputer-aided design or an algorithmic design system. In anotherembodiment, the algorithmic design system allows for the systematicvariation of post, pit, or patch configuration and spacing.

In another embodiment, writing the array pattern comprises using a highresolution electron beam lithography system. In one embodiment, writingthe array pattern comprises patterning a mask.

In one embodiment, the substrate is coated with a resist. In oneembodiment, the resist is a positive resist. In another embodiment, theresist is a negative resist.

In another embodiment, forming the post, pit, or patch comprises atechnique selected from the group consisting of liftoff, electroplating,reactive ion etching, ion milling, controlled wet etching, or acombination thereof.

In one embodiment, the method of making the device further comprisesforming microfluidic channels. In one embodiment, the method furthercomprises binding a ligand to a post, pit, or patch on the device.

Patches can be advantageous in some situations by providing a simplefabrication method. For example, patterns can be stamped using a mastertemplate (e.g., microcontact printing). Pits can be advantageous in somesituations by providing regions where the portions that bind to thesurface can be shielded from observation if desired.

Definitions

The term “post” is used herein to mean a support rising vertically froma surface.

The term “pit” is used herein to mean a structure or void penetratingvertically into a surface.

The term “patch” is used herein to mean a region on or near a surfacehaving at least one chemical difference from its surrounding.

The term “surface” is used herein to mean the outer part of anymaterial.

The term “pitch” is used herein to mean the distance between centerpoints of adjacent posts, pits, or patches.

The term “gradient” is used herein to mean a change that can be abruptor gradual.

The term “biocompatible” is used herein to mean being compatible withliving tissue by virtue of a lack of toxicity or ability to causeimmunological response.

The term “TIRF” is used herein to mean total internal reflectionfluorescence.

The term “FRET” is used herein to mean fluorescence resonance energytransfer.

The term “AFM” is used herein to mean atomic force microscopy.

The term “PMMA” is used herein to mean poly(methyl)methacrylate.

The term “resist” is used herein to mean a radiation sensitive layer.

The term “CAD” is used herein to mean computer aided design.

The term “unit cell” as used herein refers to a set of posts, pits, orpatches having a specific geometric configuration, which can include apair of posts, pits, or patches separated by a given spacing; or a smallnumber of posts, pits, or patches separated by a given spacing withfixed angles between them, so that they can be arranged as, for example,squares, rectangles, or general trapezoids.

The term “AAA” is used herein to mean ATPases associated with cellularactivity.

The term “HC” is used herein to mean heavy chain.

The term “IPA” is used herein to mean isopropanol.

The term “about” is used herein to mean approximately, roughly, around,or in the region of. When the term “about” is used in conjunction with anumerical range, it modifies that range by extending the boundariesabove and below the numerical values set forth. In general, the term“about” is used herein to modify a numerical value above and below thestated value by a variance of 20 percent up or down (higher or lower).

As used herein, the word “or” means any one member of a particular listand also includes any combination of members of that list.

Fabrication of Nanoarray Devices

The disclosed subject matter provides a coherent strategy for thepreparation of nanoscale patterns for the study of biological molecules.The methods demonstrate precision and control at the nanometer regime.

This work takes advantage of metal and semiconductor processing onscales that match the size and/or spacing of features of specificprotein or subcellular protein complexes, some of which have dimensionsof only a few nanometers. In order to create these structures, multipleapproaches can be utilized: electron beam lithography, nanoimprintinglithography, contact printing, and directed self-assembly.

For some applications described herein, hierarchical arrays of diversematerials to which specific proteins can bind, are patterned on atransparent substrate. The arrays consist of unit cells comprised ofsmall numbers (about 2-4) of metal dots separated from one another bydistances of about 10-200 nm and in some cases arranged in specificspatial configurations according to certain crystallographic spacegroups. Each array contains a sufficient number of posts within an areaof about 1-5 micrometers so as to be easily detected by TIRF microscopy.Permutations on the dot spacing and/or the precise spatial configurationof the dots in each unit cell form the next level of hierarchy in thearrays, which cover areas of several micrometers to several millimeters.Such hierarchical arrays cannot be created exclusively by chemicalself-assembly techniques (G. M. Whitesides, J. P. Matthias, and C. T.Seto, (1991) Science, 254, 1312), which are better suited toapplications requiring vast numbers of identical elements, but withlittle or no long range order and no simple way of varying theintra-unit cell spacing. Instead, more conventional “top down”lithographic techniques are used (C. R. K. Marian and D. M. Tennant(2003), J. Vac. Sci. and Technology, A21, S207), which have theflexibility to form patterns comprising many different unit cellvariations.

The required dimensions (both the feature size and spacing) of suchbioarrays are near the current limits of lithographic patterningtechniques. As an example, ultrahigh resolution electron beamlithography, with precise exposure dose control for critical features,and the use of small radius of gyration resist materials with lowtemperature processes that enhance resolution and maintain contrastextends the limits of these techniques. The patterns are transferred byliftoff and/or ion beam etching to metals for which the bindingchemistry of various biological molecules is known (A. Ulman (1996),Chem. Rev., 96, 1533 (Washington, D.C.)). For some applications, arrayscomprising diverse chemical species can be of interest. For suchapplications, multiple lithographic steps can be used with ultrahighplacement accuracy to pattern arrays of dissimilar metals (for example,gold dots followed by nickel dots could create a pattern for bindingsulfhydryl and poly-histidine tags, respectively). As another example,patterning can be achieved by scanning probe lithography, includingdip-pen, x-ray, or extreme UV lithography, and contact printing (C. R.K. Marian and D. M. Tennant (2003), J. Vac. Sci. and Technology, A21,S207).

A proposed process flow for the fabrication of hierarchical bioarrayswith molecular spacings is shown in FIG. 2. First, arrays can bedesigned using either a generic CAD package or an algorithmic designsystem, such as CADENCE, MENTOR GRAPHICS, DESIGN CAD, and the like,allowing for the systematic variation of unit cell configuration andspacing. The design data can be used as input to a high resolutionelectron beam lithography (e-beam) system, which writes each patternonto a transparent substrate coated with a radiation sensitive layer(resist). E-beam lithography can be used to pattern a mask, from whichthe array patterns are subsequently transferred by nanoimprintlithography, described below. Depending upon the details of the patterntransfer process, either a positive or negative tone resist is used.Pattern transfer can then take place via an additive process, such asliftoff or electroplating, or via a subtractive process, such asreactive ion etching, ion milling or controlled wet etching, dependingupon the selected materials. Once metal dots have been formed on thesubstrate, subsequent processing depends upon the precise application.For example, for the investigation of protein binding, microfluidicchannels can be formed, and ligands can be bound to the appropriate dotson the array. After washing and blocking the rest of the surface toprevent non-specific interactions, the binding molecules tagged with anappropriate fluorophore can be added through the flow channel, incubatedto allow binding, and washed out to measure release. A linker moleculecan be introduced which adheres to the metal by chemical bonding. Forprotein crystallization, the arrays can be used with or without furtherprocessing.

Generally, it is difficult to form features smaller than about 10 nm andarrays with center-to-center spacings less than about 50 nm usingtop-down patterning (i.e., conventional lithography) (Broers, A. N.(1988) IBM J. Research and Dev't, 32(4):502-513). However, as can beseen in FIG. 3, as early as 1981, features with nm-scale dimensions havebeen patterned by electron beam lithography (Isaacson, M. and A. Murray(1981) J. Vacuum Sci. Tech., 19(4): 1117-1120), and arrays with pitchesof about 10 nm have been realized (Langheinrich, W. and H. Beneking(1993) Jap. J. App. Physics Part 1—Reg. Papers Short Notes Rev. Papers,32(12B):6218-6223). FIG. 3(a) shows sub-5 nm lines patterned in NaClfilm by electron beam-induced vaporization as in Isaacson and Murray,1981. FIG. 3(b) shows sub-10 nm pitch lines patterned in Al-doped LiF,also as in Isaacson and Murray, 1981. In each case, exceedingly highdoses were required, and pattern transfer was problematic. Untilrecently, the key challenges in accessing the sub-10 nm regime have beenin identifying practical lithographic materials with sensitivities thatallow for manageable exposure times and are amenable to direct patterntransfer. Such materials have recently been discovered (Namatsu, H. etal. (1998) J. Vacuum Sci. Tech., 16(6):3315-3321; Rooks, M. J. and A.Aviram (1999) J. Vacuum Sci. Tech. B. 17(6):3394-3397; Yasin, S. et al.(2001) App. Physics Lett., 78(18):2760-2762), and new approaches toprocessing (Yasin, S. et al. (2001) J. Vacuum Sci. Tech. B,19(1):311-313) are also extending the resolution of commonly usedelectron beam resists, such as PMMA. The e-beam system is an FEIscanning electron microscope fitted with Nabity e-beam lithographycontrol system. Lithography can be done at energies between 1-30 keV,and the system has already demonstrated sub-10 nm patterning capability,although low probe current limits practical pattern density and coveragefor large arrays. The Leica VB-6HR is generally operated at 100 keV.This system has sufficient probe current to pattern arrays spanning manymm in a reasonable exposure time, and it has also shown sub-10 nmpatterning capability, and sub 5-nm patterning capabilities are beingpursued. FIG. 4 shows arrays of dots of hydrogen silsequioxane (HSQ), anegative tone electron beam resist. FIG. 4(a) shows ultrahigh resolutionpatterning of 6 nm HSQ dots on 100 nm pitch, while FIG. 4(b) shows a 25nm pitch n×n array. Relatively isolated features as small as 6 nm havebeen patterned, and dense arrays, with center-to-center spacing down to25 nm have already been achieved. Such high resolution is possiblebecause of the cage-like structure of the HSQ molecule (Siew, Y. K. etal. (2000) J. Electrochem. Soc., 147(1):335-339) and its small radius ofgyration (Namatsu, H. et al. (1998) J. Vacuum Sci. Tech. B,16(1):69-76). Reducing the molecular weight of the material is likely toresult in further improvements in “raw resolution.” HSQ can be easilyconverted to SiO₂ by thermal treatment or by exposure to an oxygenplasma, so that careful etching in dilute hydrogen fluoride (HF) can beused to reduce the dimensions of the dots even further. The dots can beused as a mask to transfer the pattern into an underlying film of metalor other material to which the selected proteins can bind, by ionmilling or reactive ion etching.

In addition to HSQ, resolution enhancing processes in the positive toneresist PMMA can be performed. As mentioned above, Yasin et al. (Yasin,S. et al. (2001) App. Physics Lett., 78(18):2760-2762) have demonstratedabout 5 nm patterning capability by use of a new resist developmentprocess using two nonsolvents, isopropanol and water, combined withultrasonic agitation. This new process offers superior contrast andresolution when compared to the conventional developer dilutemethyl-isobutyl-ketone (in IPA). It appears that EPA and water combineto form a co-solvent which is able to more efficiently penetrate thepolymer matrix with minimum resist swelling (which normally leads to aloss of resolution). The ultrasonic agitation induces microstreaming,yielding an efficient mechanism for providing fresh developer to thepolymer and rapid removal of dissolved material. Following Rooks et al.,(Rooks, M. J. et al. (2002) J. Vacuum Sci. Tech. B, 20(6):2937-2941),this process was improved by reducing the developer temperature,resulting in increased contrast relative to room temperature development(as well as conventional developers) (Rooks, M. J. et al. (2002) J.Vacuum Sci. Tech. B. 20(6):2937-2941). This process is employed for thefabrication of electrodes for the study of molecular electronics, anexample of which is shown in FIG. 5, with sub-5 nm spacing. FIG. 5 showsa an electrode pair for the study of transport in individual moleculesfabricated by direct-write e-beam lithography and low temperatureultrasonic development using an IPA:H₂O mixture. We are extending boththe resolution and reliability of this process by the use of lowmolecular weight PMMA. Initial experiments (Greci (2003) privatecommunication) indicate that low temperature development using anIPA/water mixture with ultrasonic agitation maintains the resistcontrast even as the molecular weight is reduced, as shown in FIG. 6.FIG. 6 shows the remaining resist thickness as a function of applieddose for a fixed development time, for four different molecular weightsof PMMA. As can be seen in FIG. 6, contrast does not degrade with areduction in molecular weight when for used for ultrasonically assistedlow temperature using an IPA:H₂O mixture. This is strikingly differentfrom what is found using conventional developers (i.e., MIBK solutions),where both contrast and resolution suffer when low Mw is used (Dobisz,E. A. et al. (2000) J. Vacuum Sci. Tech. B, 18(1): 107-111). Inaddition, scanning probe analysis of partially developed resist showsthat the low molecular weight has significantly lower surface roughnessthan the higher molecular weight material when processed using lowtemperature ultrasonic development, as shown in FIG. 7. FIG. 7 showsatomic force micrographs of partially developed PMMA of differentmolecular weights. Reduced surface roughness has been shown to correlatewith improved resolution (Namatsu, H. et al. (1998) J. Vacuum Sci. Tech.B. 16(6):3315-3321; Yasin, S. et al. (2001) J. Vacuum Sci. Tech. B.19(1):311-313), and preliminary experiments indicate that improvedresolution and linewidth control are indeed achieved with low molecularweight PMMA.

When a positive tone resist, such as PMMA, is used for the fabricationof bioarrays, pattern transfer is achieved by liftoff or by electro- andelectroless plating. The electron beam evaporation system can includesub-nanometer thickness control and liquid nitrogen-cooled stage,offering the possibility of ultrafine grain metal deposition. When anegative tone resist, such as HSQ, is used, the pattern transfer is doneby etching—reactive ion etching, ion milling, or carefully controlledwet chemical etching, depending on the materials involved.

Once dots with sub-10 nm have been formed, further dimensional reductioncan be achieved as illustrated by the thermal treatment depicted in FIG.8. After lithography and pattern transfer, each metal dot approximates acylindrical disc sitting on the substrate. Thermal treatment causes thedots to minimize their surface area, forming spheroids. Reduction of thedot thickness to a fraction of its diameter results in a significantreduction in the final dot size. For example, a dot with a diameter of 6nm and a thickness of 0.5 nm is reduced to about a 3 nm sphere. For thisapproach, precise control over film thickness and grain structure isalso important. Table I tabulates the reduction in dimensions depictedin FIG. 8. TABLE 1 Dimensional Reduction by Thermal Treatment d_(c) (nm)h (nm) d_(s) (nm) 10 1 3.9 10 0.5 3.0 8 1 3.3 8 0.5 2.6

For some applications, it is desirable to form patterns of closelyspaced posts of different materials, such as gold and nickel-NTA. Inthis case, two levels of lithography is required, with level-to-leveloverlay of about 10 nm. Such placement accuracy is achievable withe-beam lithography using careful alignment strategies (Guillorn, M. A.et al. (2000) J. Vacuum Sci. Tech. B. 18(3):1177-1181). As an example,FIG. 9 shows a set of four interdigitated metal lines in whichsuccessive pairs of alternative lines were patterned in separatelithographic exposures and metal depositions (Wind, S. J. et al. (2003)J. Vacuum Sci. Tech. B, accepted for publication). Throughhigh-resolution placement accuracy using e-beam lithography, levels Aand B depicted in FIG. 9 were patterned and processed separately. Theenlargement on the right in FIG. 9 shows better than 5 nm overlay.

FIG. 10 depicts an exemplary device fabricated by the above process.

FIG. 11 depicts an optical electron micrograph (FIG. 11(a)) and ascanning electron micrograph (FIG. 11(b)) of a prototype chip withpatterned arrays according to the some embodiments of the disclosedsubject matter. The micrographs are each about 300 microns square. Thecircular flames around each of the arrays are visible in FIG. 11(a).

FIG. 12 depicts prototype dot pair arrays fabricated by the aboveprocess.

FIG. 13 shows a prototype dot array fabricated by the above process, anddemonstrates a linear array of dot strings having specific spacings.

FIG. 14 depicts atomic force microscopy of gold-patterned prototypearrays fabricated by the above process.

Nanoimprint lithography can also be utilized. FIG. 15 depicts a thermalsystem and a photocurable system for mass-producing a device usingnanoimprint lithography. As shown, nanoimprint lithography includesimprinting and pattern transferring. Imprinting can include pressing amold onto a substrate having a layer of resist material using heat orradiation and removing the mold. Pattern transfer can include reactiveion etching to obtain a desired device in accordance with someembodiments of the disclosed subject matter. Other lithographicalternatives for patterning include PMMA, platinum, and directpatterning of self-assembled monolayers, as depicted in FIG. 16.

NANOARRAY DEVICE AND USES THEREOF

The disclosed subject matter can be utilized in a number of differentapplications. For example, nanoarray devices of the disclosed subjectmatter can be utilized as sensor devices (e.g., to detect hazardousmaterials or disease markers), as tools for biological mechanism studies(e.g., tools to study binding specificity or affinity), crystallizationtemplates (e.g., crystallization of proteins, viruses, and the like),separation devices (e.g., separation based on different structuralisomers, position isomers, functional group isomers, stereoisomers, andthe like), and the like. For example, the disclosed subject matter canbe utilized in bioinformatics, tissue engineering, portable screeningdevices, and the like.

In one embodiment of the disclosed subject matter is provided a devicecomprising (a) a substrate having a surface and (b) an ordered array ofposts, pits, or patches over the surface, wherein the posts, pits, orpatches are capable of binding a ligand, and wherein the pitch betweenadjacent posts, pits, or patches is less than about 100 nm. The deviceaccording to some embodiments of the disclosed subject matter is aflexible device that provides many new capabilities for measuring notonly the spatial dependence of binding to one ligand, but also thespatial dependence of binding to several different ligands that arepatterned on the surface. Using high throughput techniques, includingmultiple stamping techniques, several different chemicals could bepatterned on a surface; for example, gold dots followed by nickel dotscan create a pattern for binding sulfhydryl and poly-histidine tags,respectively. Stamping techniques include nanoimprint lithography andcontact printing.

In another embodiment, the disclosed subject matter provides a method torapidly measure highly specific interactions that depend upon themolecular level spacing of components in large complexes. By changingthe distance between 2-5 nm posts, pits, or patches on the nanometerscale, it is possible to create spatial arrays of ligands that match thespacing and distribution of binding sites in a binding complex. Bindingconstants can be measured directly using a microfluidics flow system andeither TIRF microscopy or ellipsometric measurements to monitor therates of binding and release of the material at known concentrations.

In a typical measurement, an array of 2-5 nm posts, pits, or patches arecreated on the template (C. R. K. Marian and D. M. Tennant (2003), J.Vac. Sci. and Technology, A21, S207). The template is then used tocreate a pattern of dots on a small area (light microscope resolutionlimits the minimum size for reliable measurements to 2-5 μm²) of a glasscoverslip. Tens to hundreds of different patterns are imprinted onadjacent regions of the coverslip to fill the 10,000 μm² viewing area ofthe microscope objective (typically a 60×, 1.45 n.a. objective capableof through objective TIRF). Once imprinted with the designated patterns,the imprinted coverslip is assembled with a microfluidics channel andthe ligands are bound to the appropriate dots on the array. Afterwashing and blocking the rest of the surface to prevent non-specificinteractions, the binding molecules tagged with an appropriatefluorophore are added through the flow channel, incubated to allowbinding, and washed out to measure release. Continuous monitoring byTIRF microscopy enables the rates of binding and release to be measuredsimultaneously for all different ligand configurations. Regions with theoptimal spacing are then correlated with the structure of the bindingcomplex, where known.

In diagnostic applications, configurations are selected that give thegreatest discrimination in binding between different complexes orbacterial species. The rate-limiting step in these measurements can bethe off rate, since binding affinities are expected to be very high forthe multiple sites. For some applications where multiple measurementsare to be made, the device is washed with mild denaturants to remove thebound complexes.

Stamping or imprinting enables the mass production of the arrays and theTIRF microscopy is automated to perform the measurements on a series ofsamples. Standard arrays are constructed for general researchapplications.

Molecular specificity with randomly oriented ligands on surfaces confersa range of binding affinities whereas this technique should providecritical spatial ordering on the molecular scale that would show a highdegree of specificity. Many cellular functions rely upon this spatialorder to confer specificity and there are an increasing number ofnanodevices that will have a similar spatial order on the nanometerlevel. Measuring spatial dependent binding is critical for defining bothcellular and artificial complexes.

The device according to some embodiments of the disclosed subject matterutilizes the spatial dependence of binding to provide furtherspecificity of binding. Several different spacings are placed on thesurface in adjacent areas in the microscope field so that relativebinding specificity is measured simultaneously. A microfluidics flowchannel over the device enables measurements to be made with as littleas 1 microliter of material. The amount of binding is determined bytotal internal reflection fluorescence microscopy and on and off ratesfrom regions with different spacings enables the proper spacing to bedetermined. For larger scale purifications and analyses, the optimalspacing is used to create an area large enough for a standardellipsometry measurement, thus enabling purification and other types ofanalyses that need larger amounts of material, and expanding the rangeof uses of the spatial binding techniques.

Specificity of binding is much greater with proper spacing of multiplesites because physical chemical calculations indicate that the bindingaffinity increases to at least the product of the affinity constants. Inaddition, other spacings on adjacent regions of the same device providecontrols for non-specific interactions. Because the volume of materialneeded for a measurement is very small (1 microliter), it is possible totest extremely small samples.

In Vitro Motor and Motility Analysis

Motor proteins such as kinesin and cytoplasmic dynein have been studiedextensively at the single molecule level. However, there are manyquestions about the structure of these proteins and the mechanism ofmotility that cannot be addressed without being able to alter the arrayof proteins that the motors move upon. For dynein, there are alsoquestions about the in vitro synthesized motor domain function that canbe addressed by arraying it on the surface. In terms of themicrotubules, different microtubule arrays have been formed but there isno current mechanism to systematically alter the array of tubulinsubunits and then to assay for binding and/or motility. Using the deviceand methods of the disclosed subject matter, the effect of tubulin dimerspacing on motor binding are determined using GFP-tubulin that is boundto a specific array of anti-GFP antibodies. A parallel analysis ofmyosin binding to arrayed actin monomers is also performed. Byengineering tubulin dimers, dimers that can be oriented on a stampedarray are developed. The ordered arrays provide one means of carryingout an in vitro motility assay and determining the effect of arraycharacteristics on motor function. This is an area of motor functionthat has not been addressed because it has not been possible withearlier technologies.

While much is known of the molecular mechanism of other classes of motorproteins, insight into the mechanism of action of the dyneins remainslimited because of their large size. The dyneins are a family ofmicrotubule based motor proteins responsible for ciliary and flagellarmotility, and play diverse roles in cell division, organelle transport,and cell movement. The dynein motor domain is unrelated to that of othercellular motors, but is a relatively divergent member of the class ofATPases associated with cellular activity. Each dynein heavy chainsubunit contains six AAA modules arrayed in a ring, from which twoprojections emerge. One is referred to as the stalk, and bindsmicrotubules to its distal tip about 10 nm from the edge of the AAAring. How ATP hydrolysis by the AAA units is converted into movement ispoorly understood. Kinesin is much better understood as a motor. Themicrotubule binding characteristics of kinesin have been extensivelystudied and provide a nice comparison for the dynein bindingcharacteristics.

Ordered nanoarrays provide an improved and in many cases unique tool toaddress a variety of important questions regarding dyneinmechanochemistry. Cytoplasmic dynein, a two-headed motor protein, wasfound to act processively in its interaction with microtubules (Wang(1995) Biophys. J., 69:2011-23). Whether the individual motor domainsare capable of sustained force production or processive behavior remainsto be determined. A single-headed form of flagellar dynein wassubsequently reported to produce force processively (Sakakibara (1999)Nature, 400:586-590). It is uncertain whether this feature is a specialevolutionary adaptation of this molecule. The dynein molecules wererandomly adsorbed to coverslips using a simple adsorption methodperformed at low protein concentrations. Although the distribution ofparticles was confirmed by TIRF microscopy using a fluorescent ATPanalogue, aggregates of dynein are not readily detected. Furthermore,this method detects fully active dynein, but so-called “dead-heads,”which fail to bind ATP but bind microtubules strongly, would be missed.

The rat cytoplasmic dynein motor domain has been expressed usingbaculovirus infection of insect cells. Full-length dynein heavy chainhas also been expressed and purified. Although it had some tendency toaggregate, full-length dynein HC was found to have limited motilityactivity (Mazumdar (1996) Proc. Natl. Acad. Sci. USA, 93:6552-6). Aconstruct of about 350 kDa corresponding to the complete motor domain,and lacking the projecting microtubule-binding region, referred to asthe stalk (Gee, (1997) Nature, 390:636-9) has also been produced. Eachof these constructs has a hexahistidine and an epitope tag, which isused for purification and/or linkage to the nanoarray supports. Bothconstructs act as unique species by sedimentation and sizingchromatography, and have high levels of ATPase activity. The motordomain construct binds microtubules efficiently, which are releasedusing ATP. These properties are strongly consistent with mechanochemicalactivity.

Additional experiments include testing for microtubule activation of theATPase activity, a further sign of motor activity. Microtubule glidingassays, in which the motor domains are adsorbed at high density tocoverslips, are performed as a more direct test for force production.The motor domains are then attached to Ni²⁺ posts. The spacing betweenposts is varied to test whether the motor domains act alone orcooperatively. If spacing of less than about 12 nm (i.e., the diameterof the motor domain) is required for full microtubule gliding activity,this suggests that two motor domains must act in concert. If the spacingmust be even shorter than this distance, it suggests that the motordomains interact through the flat top or bottom surfaces of the AAArings. A compact morphology for the two cytoplasmic dynein motor domainshas been observed in one study (Amos (1989) J. Cell Sci., 93:19-28),though no direct evidence for such an interaction between dynein motordomains has been forthcoming. If the motor domains are functional atgreater spacing, this will provide evidence that the individual domainsare functional. In order to ensure no more than one motor domain permicrotubule, short microtubules (about 1 μm) are applied to nanoarraysbearing dynein motor domains at even greater spacing.

If the motor domain fails at any spacing to support microtubule glidingactivity, this means that the peptide tag being used can be located at asuboptimal site. Versions of the motor domain with tags at each end arethen tested. Different modes of attachment are also tested, for exampleusing antibody against the epitope tag to increase the flexibility andlength of the link to the coverslip.

If motility is observed using both motor domain constructs, it is ofconsiderable interest to compare the step size obtained in each case.Based on single particle image averaging of dynein electron micrographs,it is proposed that the power stroke primarily involves a shift in theinteraction between the stem and the AAA ring, and secondarily betweenthe microtubule-binding stalk and the AAA ring. By measuring the stepsize for each construct (Gelles (1988) Nature 331:450-3; Wang (1995)Biophys J., 69:2011-23), the disclosed subject matter provides methodsfor testing directly which portion of the motor domain makes the moresignificant contribution, and, in turn, gain valuable new insight intohow the motor domain functions.

Numerous fragments of the cytoplasmic dynein heavy chain using bothbaculovirus infection of insect cells and transformation of bacterialcells for recombinant protein production have been produced to analyzedynein function. Of particular interest is the full motor domainconstruct, which contains all of the elements thought to be required forforce production. This construct is being produced in milligramquantities with either a hexahistidine or FLAG epitope tag. At least twotypes of experimental set-ups are possible using ordered nanoarrays.First, the dynein motor domain is attached to Ni²⁺ bearing dots throughthe hexahistidine tag synthesized as part of the full motor domainconstruct. Microtubules are applied to the array in the presence of ATP,and microtubule gliding motility is evaluated and quantified as afunction of dot spacing. These experiments provide new insight intowhether dynein motor domains function by a cooperative mechanism and howthe optimal spacing between motor domains compares with the spacingbetween tubulin subunits in the microtubule lattice. The nanoarrays ofdynein are also tested for their ability to seed crystallization, as afirst step toward determining the structure of the motor domain atatomic resolution.

To test for the important spacing in the substrate array, tubulin andtubulin fragments are linked to the dots within the nanoarray.Initially, tubulin dimers are linked by hexahistidine tags to Ni-NTAdots, giving a random orientation of the dimers. A similar experiment isperformed with hexahistidine actin to look for myosin binding. To lookfor more detailed aspects of the binding and the motility, an orientedarray of tubulin dimers is developed. Hexahistidine is used for onesubunit and cysteines used for the other subunit. Cysteine reacts withgold and hexahistidine reacts with Ni-NTA dots, respectively. The goldand Ni-NTA posts are successively imprinted on the substrate with adirected 5 nm displacement.

For measurement of binding and possibly mobility, the dynein motordomain is bound to latex beads or native dimers will be purified. Thebeads or dynein dimers are applied to the nanoarrays and tested forbinding or dynein-mediated movement. These experiments determine thespacing of the tubulin binding sites, step size inherent in the dyneincrossbridge cycle, and whether the motor protein can accommodate to animperfect lattice and still produce force.

Analysis of Integrin and Actin Interactions as a Function of Spacing

There are many large cytoskeletal proteins with multiple binding sitesthat are spaced by 20-100 nm (see Djinovic-Carugo, K. et al. (2002) FEBSLett., 513:119-23; Goldmann, W. H. et al. (1996) J. Muscle Res. CellMotil., 17:1-5; Liu, S. et al. (1997) Eur. J. Biochem., 243:430-6).These proteins have critical functions in cells and tissues that dependupon their specific binding to other components. We are exploring thespatial dependence of their binding interactions by measuring theirrelative interaction with different arrays of their binding partners.Theoretical analyses suggest that the proper spacing can increasebinding avidity by orders of magnitude. Nanofabricated arrays now makeit possible to measure the exact spatial dependence of the bindinginteractions. As one example, we are exploring the spatial dependence ofintegrin and actin arrays on their interactions with a variety ofbinding partners both in vivo and in vitro.

There is considerable evidence that the spacing between ligandedintegrins strongly affects binding to cytoplasmic proteins such astalin. For example, the binding of a fibronectin trimer causes specificattachment of talin1 to the cytoplasmic tail of integrin avb3. Talin1 isan anti-parallel dimer with both actin and integrin binding sites thathas an overall length of about 50 nm whereas the fibronectin-integrinbinding sites on the trimer are 40-70 nm apart. One explanation for thespecific binding of the trimer to the actin cytoskeleton is that thespacing of the liganded integrins matches the spacing of the talin1integrin binding sites. To test for this, arrays of cytoplasmic domainsof integrins with different spacings are created. If a particularspacing binds GFP-talin1 more avidly than other spacings, adetermination is made as to whether this corresponds to the full lengthof the talin1 dimer or to some other parameter of the molecule.

More specifically, recent studies indicate that the spacing of integrinsand of actin is critical for the specific binding of many proteins(Calderwood, D. A. and M. H. Ginsberg (2003) Nat. Cell Biol., 5:694-97).For example, a single trimer of fibronectin (FIG. 1(a)) forms a slipbond with the cytoskeleton (FIG. 1(b)), whereas randomly spaced monomersof fibronectin do not (Jiang, G. et al. (2003) Nature, 424:334-37). Acritical aspect of forming slip bonds is the selective binding oftalin1. Talin1 is an anti-parallel dimer with an overall length of 56 nm(Winkler, J. et al. (1997) Eur. J. Biochem., 243:430-36). Since talinhas an integrin binding site at the N-terminal end and an actin bindingsite at the C-terminus, an optimal spacing for beta integrin binding atboth sites is theoretically about 50 nm. In the fibronectin trimer, thespacing between the pairs of RGD binding sequences is maximally about 60nm (Coussen, F. et al. (2002) J. Cell Sci., 115:2581-90). The matchbetween the spacing of the binding sites on the outside of the cell andthose on the inside is very good and could be an important factor increating the specific binding complexes. Indeed, the trimer rapidlyforms a slip bond to the cytoskeleton that is broken at a force of 2pNewton whereas monomer-coated beads bind more slowly and don't showpreferential breaking at 2 pN. It is believed that the spacing of themonomeric integrins on beads varied and was often much greater than canbe spanned by talin1. The disclosed subject matter enables placingfibronectins or integrins at specific spacings on a surface. The optimalspacing can be determined by in vitro and in vivo testing.

Cells bind to extracellular matrix-coated glass differently than to thesame matrices in three dimensions, as a result of the spatialorganization of matrix subunits. We are analyzing cell spreading ondifferent arrays of fibronectin with defined spacings in the range of20-150 nm. Initially, we will prepare substrates with 30 micron squareswith a given array of fibronectin to allow cells to bind to that array.A small spacing is used and increased to determine the maximumseparation that allows binding. Pairs of gold dots (2-5 nm in diameter)are centered in 150×300 nm areas and the spacing between the dots isvaried from 25 to 150 nm. Other arrays with rows of dots spaced by20-150 nm are formed at a row-to-row spacing of 150 nm. Stamping orimprinting technology produces more closely spaced rows (giving squarearrays from 20×20 nm). The order in the arrays produces order in thecell spreading process. Cell spreading analysis enables us toquantitatively analyze the effect of order on spreading. The spreadingprocess of talin1 mutant cell lines and other cell lines are alsoinvestigated.

There are many actin binding and integrin binding proteins that have ananti-parallel dimer structure (see Dhermy, D. (1991) Biol. Cell.,71:249-54; Goldmann, W. H. et al. (1996) J. Muscle Res. Cell Motil.,17:1-5; Liu, S. et al. (2000) Eur. J. Biochem., 243:430-6). The lengthof these molecules is somewhat variable because they are often composedof repeated domains such as the spectrin domains that have flexiblelinkages between the domains. Theoretically, alpha actinin can spanabout 60 nm whereas the larger talin molecule can span over 100 nm andis reported to have 4 binding sites for beta 1 and 3 integrincytoplasmic tails. Since it is believed that talin causes a separationof the alpha and beta tails, it is likely that the isolated betacytoplasmic domains will be capable of binding talin. For in vitroexperiments, the device has smaller areas (1.5×3 μm) with the samespacings of the gold posts. This gives 100 binding pairs over that area,which can be viewed readily in TIRF.

The array of posts on a clean glass surface is prepared, (Chemiasvskayaet al., (2005), J. Vac. Sci. and Technol. B, 23:2972) and then the openareas of glass are reacted with PEG-sylanizing reagent to preventnon-specific absorption of the protein. To ensure the proper orientationof the bound proteins, the beta cytoplasmic tails are expressed inbacteria with a construct that places biotin on the amino terminus,which faces the membrane. Avidin is bound to the gold dots and then thebiotin fragments will be added. Initially, the sites are saturated withbiotin to examine the effect of spacing. Addition of a cysteine in theN-terminal region of the peptide enables us to fluorescently tag thefragment and then assay the density of bound cytoplasmic fragments inregions with single 5 nm gold dots spaced by over 0.5 micron from otherdots. Images of single fluorophores are quantified to determine theaverage number of fluorophores per dot, using the intensity and thebleaching characteristics, since bleaching of one of two fluorophorescuts the intensity in half, while single fluorophores blink out, asreadily apparent to one of ordinary skill in the art. Similarly, bindingregions are manifest as a function of incremental changes influorescence level.

EXAMPLES Example 1

As shown in FIG. 2, a CAD software, such as CADENCE from SYNOPSIS orDESIGNCAD from IMSI, is utilized to form a pattern that can be utilizedto form an array of posts. First, a thin layer of PMMA is deposited on asilicon wafer using spin casting. The pattern formed using the CADsoftware is transferred onto the PMMA layer utilizing electron beamlithography, such as a scanning electron microscope (FEI XL 30 SIRION)equipped with a NABITY NPGS pattern generator or a LEICA VB6-HR. Afterthe pattern is transferred, the PMMA layer has regions of holes ofapproximately 5 to 20 nm. A thin layer of titanium (ranging from about0.5 to 30 nms) is deposited on the surface of the wafer. Lift-off of thePMMA layer provides posts of titanium standing on the surface of thesilicon wafer. Size reduction of the titanium posts is carried out undera non-oxidizing environment at about 400° C. for about 1 hour in argonor nitrogen gas. The titanium posts are oxidized to form posts oftitanium dioxide at about 300° C. in air for a few minutes. The titaniumdioxide posts can have advantages over gold posts because titaniumdioxide does not significantly contribute to fluorescence quenching whenobserving fluorescent molecules bound on the titanium dioxide posts.

Example 2

As shown in FIG. 2, a CAD software, such as CADENCE from SYNOPSIS orDESIGNCAD from IMSI, is utilized to form a pattern that can be utilizedto form an array of pits. A thin layer of nickel is deposited on thesilicon wafer surface followed by a thin layer of gold-palladium alloy(ranging from about 0.5 to 30 nms). The gold-palladium alloy is utilizedbecause the grains of the gold-palladium alloy are significantly smallerthan the gold and provide a smoother surface. A layer of PMMA(approximately 20 to 60 nm thick) is deposited on top of thegold-palladium alloy layer using spin casting. The pattern obtainedusing the CAD software is transferred onto the PMMA layer utilizingelectron beam lithography, such as a scanning electron microscope (FEIXL 30 SIRION) equipped with a NABITY NPGS pattern generator or a LEICAVB6-HR. After pattern transfer, the PMMA layer has regions of holes ofapproximately 5 to 20 nm. Reactive ion etching or ion milling is carriedout to form pits of gold-palladium on the gold-palladium layer to formpits in the gold-palladium layer. Any PMMA material is optionallyremoved using a solvent (e.g., acetone) or by heating above thedepolymerization temperature of PMMA (e.g., 200° C.).

Example 3

As shown in FIG. 2, a CAD, such as CADENCE from SYNOPSIS or DESIGNCADfrom IMSI, is utilized to form a pattern that can be utilized to form anarray of pits. To carry out nanoimprint lithography, a master templateis formed as follows. On a silicon wafer, PMMA layer is deposited andthe pattern obtained from the CAD software is transferred onto the PMMAlayer using electron beam lithography. Then, reactive ion etching or ionmilling is carried to form posts of silicon. On a different siliconwafer, a layer of titanium is deposited and oxidized to form a layer oftitanium dioxide. On top of the titanium dioxide layer, a layer ofpolystyrene is deposited using spin casting, where the thickness of thepolystyrene layer is smaller or same as the height of the silicon postsdescribed above. The silicon wafer having the polystyrene layer isheated above the glass transition temperature of the polystyrene (e.g.,180° C.) and the wafer having silicon posts is pressed onto thepolystyrene layer and cooled down to room temperature. Once thepolystyrene has vitrified, the wafer having silicon posts is removedleaving a pattern of holes in the polystyrene layer. Then, ion millingis carried out to form pits in the titanium dioxide layer. Any remainingpolystyrene is removed using solvents (e.g., toluene).

Example 4

As shown in FIG. 2, a CAD software, such as CADENCE from SYNOPSIS orDESIGNCAD from IMSI, is utilized to form a pattern that can be utilizedto form an array of patches. A thin layer of nickel is deposited on thesilicon wafer surface followed by a thin layer of gold (ranging fromabout 0.5 to 30 nms). A layer of PMMA (about 20 to 60 nm thick) isdeposited on top of the gold layer using spin casting. The patternobtained using the CAD software is transferred onto the PMMA layerutilizing electron beam lithography, such as a scanning electronmicroscope (FEI XL 30 SIRION) equipped with a NABITY NPGS patterngenerator or a LEICA VB6-HR. After pattern transfer, the PMMA layer hasregions of holes, exposing small areas of gold of about 5-20 nm in size.A solution or vapor of thiol-functionalized molecules is brought incontact with the exposed gold regions to form a monolayer of molecules.The PMMA layer is removed using a solvent (e.g., acetone) producingpatches of molecules on the gold surface.

While the disclosed subject matter has been described in detail withreference to some embodiments thereof, it will be understood that thedisclosed subject matter is not limited to these embodiments. Indeed,modifications and variations are within the spirit and scope of thatwhich is described and claimed.

1. A device for measuring nanometer level binding reactions, the devicecomprising: (a) a surface; and (b) an ordered array of posts, pits, orpatches on the surface, wherein the posts, pits, or patches are capableof binding a protein or small molecule ligand, wherein the pitch betweenadjacent posts, pits, or patches is less than about 100 nm, and whereineach post consists essentially of titanium dioxide or gold-palladiumalloy.
 2. The device of claim 1, wherein the device comprises a gradualchange of pitch that varies from about 5 nm to about 100 nm.
 3. Thedevice of claim 1, wherein the post, pit, or patch has affixed theretoat least one protein molecule.
 4. The device of claim 3, wherein one ofthe post, pit, or patch has affixed thereto exactly one proteinmolecule.
 5. The device of claim 1, wherein the protein moleculecomprises at least a portion of a dynein heavy chain, a tubulin, akinesin, a myosin, a cytoplasmic domain of an integrin, an actin, anextracellular matrix protein, a fibronectin, a collagen, a laminin, aDNA solenoid, a DNA, a histone-DNA complex, an RNA, an RNA-proteincomplexes, a bacterial coat proteins, an antibody, a lectin, an avidin,or any combination thereof.
 6. The device of claim 1, wherein theprotein molecule comprises a full-length dynein heavy chain.
 7. Thedevice of claim 1, wherein the protein molecule comprises a dynein motordomain.
 8. The device of claim 7, wherein the protein molecule comprisesan N-terminal portion of the dynein motor domain.
 9. A method formeasuring a nanometer level binding reaction, the method comprising (a)providing a device comprising a surface; the surface comprising anordered array of posts, pits, or patches, wherein the pitch betweenadjacent posts, pits, or patches is less than about 100 nm, wherein eachpost, pit, or patch is coated with ligand, and wherein each postconsists essentially of titanium dioxide or gold-palladium alloy; (b)contacting the surface of the device with a sample; and (c) determiningwhether or not an analyte from the sample interacts or binds to aligand-coated post, pit, or patch to measure the nanometer level bindingreaction.
 10. The method of claim 9, further comprising isolating theanalyte from the sample.
 11. The method of claim 9, wherein the devicecomprises a gradual change of pitch that varies from about 5 nm to about100 nm.
 12. The method of claim 9, wherein the post, pit, or patch hasaffixed thereto at least one protein molecule.
 13. The method of claim12, wherein one of the post, pit, or patch has affixed thereto exactlyone protein molecule.
 14. The method of claim 12, wherein the proteinmolecule comprises at least a portion of a dynein heavy chain, atubulin, a kinesin, a myosin, a cytoplasmic domain of an integrin, anactin, an extracellular matrix protein, a fibronectin, a collagen, alaminin, a DNA solenoids, a DNA, histone-DNA complex, an RNA, anRNA-protein complex, a bacterial coat protein, an antibody, a lectin, anavidin, or any combination thereof.
 15. The method of claim 12, whereinthe protein molecule comprises a full-length dynein heavy chain.
 16. Themethod of claim 12, wherein the protein molecule comprises a dyneinmotor domain.
 17. The method of claim 16, wherein the protein moleculecomprises an N-terminal portion of the dynein motor domain.